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Site-Directed Mutagenesis
Site-directed mutagenesis allows for the introduction of changes in DNA to study structural and/or functional properties of proteins, RNA, or DNA. Primers are designed long enough on each end bind to the target sequence, with the selected deletion, insertion, or substitution in the middle of the primer. Through PCR, the DNA is denatured by heating, thereby splitting the strands apart. The special primers with the mutation in the middle bind to the denatured DNA. During elongation, the polymerase binds and adds dNTPs. Cycle time amplifies the DNA with the selected mutation. Method Origins/Development Originally, non-specific mutagenesis was conducted with chemicals or radiation treatment. As time progressed, nucleotide analogs began to be used to obtain point mutations. Directed mutagenesis was first described by Dr. Charles Weissmann and collegues using bacteriophage Q RNA. In 1973, a G to A substitution was introduced into the bacteriophage. ATP and GTP were used as the substrates. CMP, or cytidine monophosphate, is a nucleotide monomer of RNA. Weissmann et al used a mutated nucleotide analog of CMP, N4-hydroxyl CMP to introduce the mutation in the bacteriophage. Synthesis with ATP and GTP allowed for incorporation of the mutated analog (Flavell et al., 1973). Procedure NEB Q5 There are several mutagenesis kits on the market, but this article will focus on New England Biolab's Q5 Site-Directed Mutagenesis kit for simplicity. Tools: NEBaseChanger Primers can be designed specially for the NEB Q5 mutagenesis kit using the NEBaseChanger tool provided by the company. Polymerase/ Hot-start high fidelity master mix Fidelity is measured by the enzyme’s ability to read the template, properly select the right nucleoside triphosphate, and correctly insert the right nucleotide at the 3’ end. Some DNA polymerases also have exonuclease activity, which helps correct mistakes by cutting out incorrect nucleotides with subsequent replacement of a correct nucleotide. The hot-start high fidelity mix and primers are essential for amplifying certain DNA with a insertion, deletion, or substitution. KLD: Kinase, Ligase, and Dpn1 Kinase: The kinase in the KLD mixture phosphorylates the amplified DNA. Phosphorylation is necessary for subsequent ligation since ligases need a phosphate in the 5’ position of the DNA. Ligase: Responsible for re-circularization. Needs a phosphorylation in the 5' position in order to work effectively. DpnI: a type II restriction enzyme, particularly a restriction endonuclease in that it cleaves phosphodiester bonds within a string of nucleotides. This enzyme identifies DNA that has been methylated and cleaves it. The enzyme is classified as “restriction” because it is only able to cut at certain sequences of nucleotides in the DNA (Restriction enzyme Wiki). Dpn1 is essential for removing template DNA. Transformation, Selection, and Sequencing Competent E. coli, which have weakened cell membranes, are used to enhance the chance of DNA incorporation during transformation of the plasmid. Transformations are plated onto agar containing an antibiotic specific to the resistance the DNA plasmid encodes. Only colonies that had plasmid uptake will grow, which are then selected, extracted of DNA, and sent for sequencing to determine if the mutagenesis was successful. Usefulness to Genetics Example: Many studies that revolve around post-translational modifications make use of site-directed mutagenesis to study a particular modification's function. For example, if a particular known phosphorylation site on a serine, threonine, or tyrosine is thought to be important, the site can be mutated to phenylalanine to create a non-phosphorylatable mutant. Functional assays can be used to compare the wildtype to the mutant. Another example: When studying a particular kinase, amino acids in the active sequence of the enzyme can be mutated to render the kinase inactive. This mutant can be compared to the wildtype to see if there is a difference in function. References Flavell RA, Sabo DL, Bandle EF, and Weissmann C. Site-directed mutagenesis: generation of an extracistonic mutation in bateriophage Q RNA. J Mol Biol 1974 (89): 255-272. Muller W, Weber H, Meyer F, and Weissmann C. Site-directed mutagenesis in DNA: generation of point mutations in cloned globin complementary DNA at the positions corresponding to amino acids 121 to 123. J Mol Biol 1978 (124): 343-358. New England Biolabs Restriction Enzyme Wiki